Cardiomyopathy is a group of disorder with genetic heterogeneity and variable expression. To develop effective therapy, it is vital to identify both causative genes and genetic modifiers. This proposal aims to establish a novel adult zebrafish-based screening approach that allows systematically identification of genetic modifiers of cardiomyopathy. Having generated a Doxorubicin-induced cardiomyopathy model and developed an efficient method to generate cardiac mutants, we tested a forward genetic screening approach for this purpose. A pilot screen of >500 gene-breaking transposon (GBT) mutants identified four mutant lines that modified DOX-induced cardiomyopathy. Three affected genes, including sorbin and SH3 domain containing 2b (sorbs2b), anoctamin 5a (ano5a) and retinoid X receptor, alpha a (rxraa), have been previously linked to cardiomyopathy; while the fourth gene, Dnaj (Hsp40) homolog, subfamily B, member b (dnajb6b), is a new cardiac gene, demonstrating the capacity of our screening method to discover both known and new cardiomyopathy genes. Before we scale up the screen, we consider it as pivotal to determine whether and how these zebrafish mutants facilitate our understanding of human cardiomyopathy. By focusing on dnajb6b, the new cardiac gene, and sorbs2b, the gene with newly defined non-cardiomyocyte expression, we propose to test the central hypothesis of this proposal predicting that novel cardiomyopathy modifying genes can be identified via a mutagenesis screen approach, which can be further studied to profile their modifying effects on different genetic types of cardiomyopathy and to develop targeted therapy. The proposal is divided into the following three specific aims. In Specific Aim 1, we will determine the modifying effects of dnajb6b and sorbs2b on different types of heritable cardiomyopathy. For this purpose, we have generated three heritable cardiomyopathy models including bag3 KO, mBAG3 OE and lmna KO. In Specific Aim 2, we will elucidate the modifying effects of dnajb6b and sorbs2b via mechanistic studies and to seek therapeutics. We will examine whether UPR inhibition ameliorates the modifying effects of dnajb6b and/or sorbs2b. In specific Aim 3, we will validate the modifying effects of human DNAJB6 and SORBS2 variants and the targeted therapeutics in both fish and mouse models. Completion of these aims are expected to demonstrate the feasibility of zebrafish to model different types of cardiomyopathy, to identify modifying genes, t elucidate the modifying effects of these genes on different type of cardiomyopathies, and to discover targeted therapeutics. If successful, our strategy shall greatly advance prognostic test development, risk stratification, and personalized therapy for cardiomyopathy.